We propose an alternate minimization algorithm for estimating the point-spread function (PSF) of a confocal laser scanning microscope and the specimen fluorescence distribution. A three-dimensional separable Gaussian model is used to restrict the PSF solution space and a constraint on the specimen is used so as to favor the stabilization and convergence of the algorithm. The results obtained from the simulation show that the PSF can be estimated to a high degree of accuracy, and those on real data show better deconvolution as compared to a full theoretical PSF model.

Confocal laser scanning microscopy is a powerful technique for studying
biological specimens in three dimensions (3D) by optical sectioning. It permits
to visualize images of live specimens non-invasively with a resolution of few
hundred nanometers. Although ubiquitous, there are uncertainties in the
observation process. As the system’s impulse response, or point-spread function
(PSF), is dependent on both the specimen and imaging conditions, it should be
estimated from the observed images in addition to the specimen. This problem is
ill-posed, under-determined. To obtain a solution, it is necessary to insert some
knowledge in the form of a priori and adopt a Bayesian approach. The state of
the art deconvolution and blind deconvolution algorithms are reviewed within a
Bayesian framework. In the first part, we recognize that the diffraction-limited
nature of the objective lens and the intrinsic noise are the primary distortions
that affect specimen images. An alternative minimization (AM) approach
restores the lost frequencies beyond the diffraction limit by using total variation
regularization on the object, and a spatial constraint on the PSF. Additionally,
some methods are proposed to ensure positivity of estimated intensities, to
conserve the object’s flux, and to well handle the regularization parameter.
When imaging thick specimens, the phase of the pupil function due to spherical
aberration (SA) cannot be ignored. It is shown to be dependent on the refractive
index mismatch between the object and the objective immersion medium, and
the depth under the cover slip. The imaging parameters and the object’s original
intensity distribution are recovered by modifying the AM algorithm. Due to
the incoherent nature of the light in fluorescence microscopy, it is possible to
retrieve the phase from the observed intensities by using a model derived from
geometrical optics. This was verified on the simulated data. This method could
also be extended to restore specimens affected by SA. As the PSF is space varying,
a piecewise convolution model is proposed, and the PSF approximated so that,
apart from the specimen, it is sufficient to estimated only one free parameter.

In this paper, we present an approach to calculate the wavefront in
the back pupil plane of an objective in a fluorescent MACROscope.
We use the three-dimensional image of a fluorescent bead because it
contains potential pupil information in the ‘far’ out-of-focus planes
for sensing the wavefront at the back focal plane of the objective.
Wavefront sensing by phase retrieval technique is needed for several
reasons. Firstly, the point-spread function of the imaging system
can be calculated from the estimated pupil phase and used for image
restoration. Secondly, the aberrations in the optics of the objective
can be determined by studying this phase. Finally, the estimated
wavefront can be used to correct the aberrated optical path with-
out a wavefront sensor. In this paper, we estimate the wavefront of
a MACROscope optical system by using Bayesian inferencing and
derive the Gerchberg-Saxton algorithm as a special case.

In this paper, we model the point-spread function (PSF) of a fluorescence MACROscope with a field aberration. The MACROscope is an imaging arrangement that is designed to directly study small and large specimen preparations without physically sectioning them. However, due to the different optical components of the MACROscope, it cannot achieve the condition of lateral spatial invariance for all magnifications. For example, under low zoom settings, this field aberration becomes prominent, the PSF varies in the lateral field, and is proportional to the distance from the center of the field. On the other hand, for larger zooms, these aberrations become gradually absent. A computational approach to correct this aberration often relies on an accurate knowledge of the PSF. The PSF can be defined either theoretically using a scalar diffraction model or empirically by acquiring a three-dimensional image of a fluorescent bead that approximates a point source. The experimental PSF is difficult to obtain and can change with slight deviations from the physical conditions. In this paper, we model the PSF using the scalar diffraction approach, and the pupil function is modeled by chopping it. By comparing our modeled PSF with an experimentally obtained PSF, we validate our hypothesis that the spatial variance is caused by two limiting optical apertures brought together on different conjugate planes.

3 - Point-spread function retrieval for fluorescence microscopy.P. Pankajakshan et L. Blanc-Féraud et Z. Kam et J. Zerubia. Dans Proc. IEEE International Symposium on Biomedical Imaging (ISBI), Publ. IEEE, Org. IEEE, Boston, USA, juin 2009.Mots-clés : fluorescence microscopy, point spread function, Algorithme EM, Deconvolution.Copyright : Copyright 2009 IEEE. Published in the 2009 International Symposium on Biomedical Imaging: From Nano to Macro (ISBI 2009), scheduled for June 28 - July 1, 2009 in Boston, Massachusetts, U.S.A. Personal use of this material is permitted. However, permission to reprint/republish this material for advertising or promotional purposes or for creating new collective works for resale or redistribution to servers or lists, or to reuse any copyrighted component of this work in other works, must be obtained from the IEEE. Contact: Manager, Copyrights and Permissions / IEEE Service Center / 445 Hoes Lane / P.O. Box 1331 / Piscataway, NJ 08855-1331, USA. Telephone: + Intl. 908-562-3966.

In this paper we propose a method for retrieving the Point-Spread Function (PSF) of an imaging system given the observed images of fluorescent microspheres. Theoretically calculated PSFs often lack the experimental or microscope specific signatures while empirically obtained data are either over sized or (and) too noisy. The effect of noise and the influence of the microsphere size can be mitigated from the experimental data by using a Maximum Likelihood Expectation Maximization (MLEM) algorithm. The true experimental parameters can then be estimated by fitting the result to a model based on the scalar diffraction theory. The algorithm was tested on some simulated data and the results obtained validate the usefulness of the approach for retrieving the PSF from measured data.

Optical Sections of biological samples obtained from a fluorescence Confocal Laser Scanning Microscopes (CLSM) are often degraded by out-of-focus blur and photon counting noise. Such physical constraints on the observation are a result of the diffraction-limited nature of the optical system, and the reduced amount of light detected by the photomultiplier respectively. Hence, the image stacks can benefit from postprocessing restoration methods based on deconvolution. The parameters of the acquisition system’s Point Spread Function (PSF) may vary during the course of experimentation, and so they have to be estimated directly from the observation data. We describe here an alternate minimization algorithm for the simultaneous blind estimation of the specimen 3D distribution of fluorescent sources and the PSF. Experimental results on real data show that the algorithm provides very good deconvolution results in comparison to theoretical microscope PSF models.

In this paper, we propose a method for the
iterative restoration of fluorescence Confocal Laser Scanning
Microscopic (CLSM) images and parametric estimation of the
acquisition system’s Point Spread Function (PSF). The CLSM is
an optical fluorescence microscope that scans a specimen in 3D
and uses a pinhole to reject most of the out-of-focus light. However,
the quality of the images suffers from two basic physical
limitations. The diffraction-limited nature of the optical system,
and the reduced amount of light detected by the photomultiplier
cause blur and photon counting noise respectively. These images
can hence benefit from post-processing restoration methods
based on deconvolution. An efficient method for parametric
blind image deconvolution involves the simultaneous estimation
of the specimen 3D distribution of fluorescent sources and
the microscope PSF. By using a model for the microscope
image acquisition physical process, we reduce the number of
free parameters describing the PSF and introduce constraints.
The parameters of the PSF may vary during the course of
experimentation, and so they have to be estimated directly from
the observed data. A priori model of the specimen is further
applied to stabilize the alternate minimization algorithm and to
converge to the solutions.

In this research report, we recall briefly how the diffraction-limited nature of an optical microscope's objective, and the intrinsic noise can affect the observed images' resolution. A blind deconvolution algorithm can restore the lost frequencies beyond the diffraction limit. However, under other imaging conditions, the approximation of aberration-free imaging, is not applicable, and the phase aberrations of the emerging wavefront from a specimen immersion medium cannot be ignored any more. We show that an object's location and its original intensity distribution can be recovered by retrieving the refracted wavefront's phase from the observed intensity images. We demonstrate this by retrieving the point-spread function from an imaged microsphere. The noise and the influence of the microsphere size can be mitigated and sometimes completely removed from the observed images by using a maximum a posteriori estimate. However, due to the incoherent nature of the acquisition system, phase retrieval from the observed intensities will be possible only if the phase is constrained. We have used geometrical optics to model the phase of the refracted wavefront, and tested the algorithm on some simulated images.

We propose a method for the iterative restoration of fluorescence Confocal Laser Scanning Microscope (CLSM) images with parametric estimation of the acquisition system’s Point Spread Function (PSF). The CLSM is an optical fluorescence microscope that scans a specimen in 3D and uses a pinhole to reject most of the out-of-focus light. However, the quality of the image suffers from two primary physical limitations. The first is due to the diffraction-limited nature of the optical system and the second is due to the reduced amount of light detected by the photomultiplier tube (PMT). These limitations cause blur and photon counting noise respectively. The images can hence benefit from post-processing restoration methods based on deconvolution. An efficient method for parametric blind image deconvolution involves the simultaneous estimation of the specimen 3D distribution of fluorescent sources and the microscope PSF. By using a model for the microscope image acquisition physical process, we reduce the number of free parameters describing the PSF and introduce constraints. The parameters of the PSF may vary during the course of experimentation, and so they have to be estimated directly from the observation data. We also introduce a priori knowledge of the specimen that permits stabilization of the estimation process and favorizes the convergence. Experiments on simulated data show that the PSF could be estimatedwith a higher degree of accuracy and those done on real data show very good deconvolution results in comparison to the theoretical microscope PSF model.